Enhancing prime editing via inhibition of mismatch repair pathway

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Background In the recent issue of Cell [1], Chen et al. from David Liu laboratory discovered that inhibition of DNA mismatch repair (MMR) significantly enhanced the efficiency of prime editing while abated the frequency of unintended indels. Prime editing (PE) is currently the most precise and versatile genome editing technique that allow all kinds of base conversions and small fragment deletion or insertion in the genome [2], but its efficiency is generally lower than that of other Cas9 derived editing tools, such as nuclease Cas9 and base editing [3]. Prime editing was also invented in David Liu laboratory by fusing MMLVreverse transcriptase (RT) to Cas9 nickase. Noteworthy, since the Nobel-winning work demonstrated that Cas9 system is reprogrammable for new target [4], it was soon widely used in the biomedical research because of its superiority over former reprogrammable nucleases. Basically, the Cas9 system consists two effectors, the Cas9 protein and its guide RNAs, crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA), which can be engineered into one single guide RNA (sgRNA). Under the guidance of sgRNA, wildtype Cas9 protein recognizes its target DNAs and produces double strand breaks. Mutant Cas9 protein with impaired nuclease activity can still bind the targets, which can be used as a platform to recruit other effectors to perform localized DNA manipulations. Cas9 disassociates the non-target strand (NTS) of sgRNA from its target strand (TS) to form a R-loop structure and the disassociated NTS is exposed outside complex and therefore can serve as a substrate for enzymes preferring ssDNAs. This feature was well utilized in designing base editors [3], where a cytosine or adenine deaminase was fused with Cas9 nickase that specifically cleaves the TS. By deaminating the cytosines or adenines in the exposed NTS, these editors converted them into uracil and hypoxanthine respectively, which were then converted into thymine and guanine respectively with the assistance of endogenous repair mechanisms. Although effective, the accuracy and versatility of base editors are limited in that they non-selectively converted their substrate bases within the editing window and they mainly perform base transitions. To resolve these issues, David Liu laboratory developed prime editors, which utilize Cas9 nickase to cleave NTS, prime editing sgRNA (pegRNA) to bind the TS, and RT to reverse transcribe the 3’ end of NTS according to the information encoded by pegRNA. These concerted actions ultimately produced a 3’ flap with intended edits, inducing endogenous DNA repair mechanisms to integrate the edits into the genome. Armed with welldesigned pegRNAs, PE enables virtually all types of editing, including point mutations, deletions and insertions. However, the efficiency of PE is generally lower than that of other Cas9 based tools (Fig. 1a). In the recent Cell paper, David Liu laboratory discovered a practical solution to enhance the efficiency of PE The authors conducted a pooled CRISPRi screening with sgRNAs targeting genes involved in DNA repair and identified that interfering MMR genes enhanced both the efficiency and accuracy of prime editing. In their screening system (Fig. 1b), named Repair-seq, Streptococcus pyogenes Cas9 (SpCas9) derived transcriptional repressor (dCas9–KRAB) was used to couple with pooled suppressor sgRNAs to inhibit gene expression and Staphylococcus Open Access Molecular Biomedicine


Background
In the recent issue of Cell [1], Chen et al. from David Liu laboratory discovered that inhibition of DNA mismatch repair (MMR) significantly enhanced the efficiency of prime editing while abated the frequency of unintended indels. Prime editing (PE) is currently the most precise and versatile genome editing technique that allow all kinds of base conversions and small fragment deletion or insertion in the genome [2], but its efficiency is generally lower than that of other Cas9 derived editing tools, such as nuclease Cas9 and base editing [3].
Prime editing was also invented in David Liu laboratory by fusing MMLV-reverse transcriptase (RT) to Cas9 nickase. Noteworthy, since the Nobel-winning work demonstrated that Cas9 system is reprogrammable for new target [4], it was soon widely used in the biomedical research because of its superiority over former reprogrammable nucleases. Basically, the Cas9 system consists two effectors, the Cas9 protein and its guide RNAs, crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA), which can be engineered into one single guide RNA (sgRNA). Under the guidance of sgRNA, wildtype Cas9 protein recognizes its target DNAs and produces double strand breaks. Mutant Cas9 protein with impaired nuclease activity can still bind the targets, which can be used as a platform to recruit other effectors to perform localized DNA manipulations. Cas9 disassociates the non-target strand (NTS) of sgRNA from its target strand (TS) to form a R-loop structure and the disassociated NTS is exposed outside complex and therefore can serve as a substrate for enzymes preferring ssDNAs. This feature was well utilized in designing base editors [3], where a cytosine or adenine deaminase was fused with Cas9 nickase that specifically cleaves the TS. By deaminating the cytosines or adenines in the exposed NTS, these editors converted them into uracil and hypoxanthine respectively, which were then converted into thymine and guanine respectively with the assistance of endogenous repair mechanisms. Although effective, the accuracy and versatility of base editors are limited in that they non-selectively converted their substrate bases within the editing window and they mainly perform base transitions.
To resolve these issues, David Liu laboratory developed prime editors, which utilize Cas9 nickase to cleave NTS, prime editing sgRNA (pegRNA) to bind the TS, and RT to reverse transcribe the 3' end of NTS according to the information encoded by pegRNA. These concerted actions ultimately produced a 3' flap with intended edits, inducing endogenous DNA repair mechanisms to integrate the edits into the genome. Armed with welldesigned pegRNAs, PE enables virtually all types of editing, including point mutations, deletions and insertions. However, the efficiency of PE is generally lower than that of other Cas9 based tools (Fig. 1a).
In the recent Cell paper, David Liu laboratory discovered a practical solution to enhance the efficiency of PE The authors conducted a pooled CRISPRi screening with sgRNAs targeting genes involved in DNA repair and identified that interfering MMR genes enhanced both the efficiency and accuracy of prime editing. In their screening system (Fig. 1b)  to explain the effect (Fig. 1c), in which MMR recognized the intermediate heteroduplex that was formed by the pairing of PE-extended 3' flap and TS. Because the 3' flap was accompanied with a nick, it tended to be excised out of the edited strand by MMR and the original, unedited TS was kept intact. Interestingly, the level of enhancement by MMR inhibition was dependent on cell types, which might due to varied MMR activities in these cells, indicating that the effect of MMR inhibition on PE depends on the state of MMR of the target cells.
The level of enhancement seemed also correlated with the types of PE, because it declined as the length of the indel loops increased, which might possibly due to the limitations of MMR in recognizing unpaired regions (1-13nt MLH1dn), which showed enhanced editing efficiency and reduced indels across a variety of cell types. Importantly, no obvious microsatellite instability was observed, suggesting that transient action of PE4 or PE5 is relative safe. However, this observation did not rule out the possibility of increasing general mutation rate, which deserves carefully examinations. In summary, Chen et al. have demonstrated that inhibition of MMR enhanced the efficiency of PE. The authors provided systematic investigation and detailed evidence to demonstrate the effects of MMR pathway on the outcomes of prime editing. Based on these findings, they also developed genetic encoded and high-efficient prime editors, PE4 and PE5, which shall enhance the potential of this technology for clinical translation.